Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 93 cooking and aroma, but these are time-consuming or unreliable. Microsatellites or simple sequence repeats (SSR) are molecular markers used for identification (2–4). Generally, interpretation of SSR data can be difficult, especially for an admixture of more than 3 individuals. In addition, fragment sizing requires a high degree of accuracy. Consequently, this method can be very time and resource-consuming, especially when performing simplex analyses using conventional gel electrophoresis. The QIAxcel Advanced capillary electrophoresis system can replace conventional gel electrophoresis with an automated process that minimizes manual intervention and errors. Analysis is fast (96 samples in 1 h 30 min) and inexpensive. Additionally, the QIAxcel ScreenGel® software estimates the sizes and areas of the fragments analyzed, rendering complex analysis software unnecessary because interpretation can be done directly with ScreenGel and the Excel® spreadsheets it produces. Using the QIAxcel DNA High Resolution DNA Kit makes an accuracy of 3–5 bp possible, which is crucial for correct sizing of SSRs. We have developed an SSR-PCR protocol for routine analysis of Basmati rice using the QIAxcel Advanced System, where all Basmati as well as other types of rice can be identified and quantified as DNA percentage of the Basmati variety. Materials and methods Grains from a range of Basmati and non-Basmati rice samples were selected and homogenized. Homogenized samples were mixed with lysis buffer (Qiagen ATL), heated to 65°C, and treated mechanically by agitation at 1400 rpm. DNA was purified with the QIAsymphony® DSP DNA Mini Kit. DNA amplification by duplex PCR was performed with a set of 8 SSR markers (RM1 + RM72, RM44 + RM55, RM202 + RM241 and RM171 + RM348) using the QIAGEN Multiplex PCR Kit. For some Basmati rice, 2 additional SSR markers were used for more accurate discrimination (data not shown). Size range of the PCR products was 80–400 bp (Figure 1). Capillary electrophoresis was carried out using the QIAxcel DNA High Resolution Kit, QX Alignment Marker 15 bp/600 bp, and QX DNA Size Marker 25–500 bp v2.0. QIAxcel ScreenGel software was used to estimate fragment size and percentage of target rice in the analyzed samples. The type of rice was identified using data from the Excel spreadsheets produced by the software. Results and discussion Identification Analysis of different types of Basmati rice at 8 loci demonstrated that the rice varieties could be identified with high discriminatory power. Results showed characteristic, stable and reproducible DNA fragment profiles for each rice variety (Figure 1). Reproducibility and stability were assessed by repeating the analysis 12 times with a Taraori Basmati rice sample (Figure 2). Quantification When non-Basmati rice was detected in a sample, the quantity of Basmati rice DNA was presented as a percentage (Figure 3). This method is based on the comparison of the allele profiles of a known pure-grain sample and the unknown, tested sample. If a new peak was detected above a given threshold level, all possible profiles of all loci were identified. The area under the peak of every marker was calculated as a percentage. The average of all values was considered the quantity of Basmati rice DNA in the tested sample (Figure 4). The overall uncertainty of the analysis was 5.2%. The uncertainty when over 95% of the mixture was Basmati rice was 1.9%, which complies with EU regulations. The quantification had an accuracy of 0.2% and a dispersion of 2.0% based on 133 measurements made on 37 different mixtures of Basmati and non-Basmati varieties.

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